Human marrow stromal cells (hMSCs) are multipotential adult stem cells that contribute to the regeneration of tissues such as bone, cartilage, fat, and muscle (1997, Friedenstein, et al., Exp. Hematol. 4(5):267-274; 1997, Prockop, D J, Science, 276(5309):71-74; 1999, Pittenger, et al., Science, 284(5411):143-147; 1998, Ferrari et al., Science, 279(5356): 1528-1530).
The recent discovery of stem cell populations in the central nervous system (CNS) has generated intense interest, since the brain has long been regarded as incapable of regeneration (Reynolds and Weiss, 1992, Science 255:1707-1710; Richards et al., 1992, Proc. Natl. Acad. Sci. USA 89:8591-8595; Morshead et al., 1994, Neuron 13:1071-1082). Neural stem cells (NSCs) are capable of undergoing expansion and differentiating into neurons, astrocytes and oligodendrocytes in vitro (Reynolds and Weiss, 1992, Science 255:1707-1710; Johansson et al., 1999, Cell 96:25-34; Gage et al., 1995, Annu. Rev. Neurosci. 18:159-192; Vescovi et al., 1993, Neuron 11:951-966). NSCs back transplanted into the adult rodent brain survive and differentiate into neurons and glia, raising the possibility of therapeutic potential (Lundberg et al., 1997, Exp. Neurol. 145:342-360; Lundberg et al., 1996, Brain Res. 737:295-300; Renfranz et al., 1991, Cell 66:713-729; Flax et al., 1998, Nature Biotech. 16:1033-1039; Gage et al., 1995, Proc. Natl. Acad. Sci. USA 92:11879-11883; Svendsen et al., 1997, Exp. Neurol. 148:135-146). However, the inaccessibility of NSC sources deep in the brain severely limits clinical utility. The recent report demonstrating that NSCs can generate hematopoietic cells in vivo suggests that hematopoietic stem cell populations may be less restricted than previously thought (Bjornson, 1999, Science 283:534-537).
Recent data suggest that MSCs can also be induced to differentiate into neural cells in vivo. It has been found that hMSCs integrate and migrate along the known pathway for the migration of neural stem cells after being infused into rat brain (Azizi, et al., 1998, PNAS, 95(7):3908-3913). Other data demonstrate that mouse MSCs (mMSCs) labeled with BrdU migrate to both forebrain and cerebellum without disruption of normal brain structure when injected into the lateral ventricle of a neonatal mouse (Kopen, et al., 1999, PNAS, 96(19):10711-10716). Some of the mMSCs differentiated into cells that had astrocyte morphology and expressed the astrocyte-specific protein glial fibrillary acid protein (GFAP). Further, some of the mMSCs appeared in the olfactory bulb and the internal granular layer of the cerebellum, both of which are neuron-rich regions. Finally, the Kopen study also demonstrated that some BrdU-labeled mMSCs found in the reticular formation of the brain stem were positive for neurofilament.
Other investigations report conditions under which MSCs may be differentiated in culture into neural-like cells. Woodbury et al. demonstrate that cells may be differentiated either by serum withdrawal and exposure to beta-mercaptoethanol (BME), or by treatment of the MSCs with butylated hydroxytoluene (BHT) and dimethylsulfoxide (DMSO) (Woodbury et al., 2000, J. Neurosci. Res., 61(4):364-370). Others report that MSCs may be differentiated into neural-like cells by treatment with epidermal growth factor (EGF) followed by brain derived growth factor (BDGF), or by co-culture with a suspension of rat or mouse midbrain cells (Sanchez-Ramos et al., 2000, Exp. Neurol., 164(2):247-256).
However, until the present invention, a need has existed to elucidate the early steps of neural differentiation so that, cells at different early stages of differentiation may be identical and used in therapy. The present invention fulfills this need.